Embryonic stem (ES) and carcinoma (EC) cells undergo differentiation in culture when treated with retinoic acid. The majority of the ES cells differentiate into cells with properties that resemble the primitive endoderm of early embryos. Retinoic acid-induced primitive endoderm differentiation leads to suppression of cell growth, which we found to result from restriction of active MAPK from the nucleus and to require an intact cytoskeleton. In previous studies, we have found that in differentiated cells, activated phospho-MAPK remains principally cytoplasmic rather than entering the nucleus, in contrast to most cultured cells, in which stimulation of cells by serum mitogens results in activated MAPK readily entering the nucleus. The localization of activated MAPK correlates with the degree of phosphorylation of nuclear and cytoplasmic MAPK substrates, such as Elk1 and cPLA2, respectively. The current proposal is to investigate a potential mechanism that accounts for the cytoplasmic retention of activated MAPK in differentiated ES cells. In preliminary studies, we found that endoderm differentiation of ES cells dramatically increases expression of the nuclear envelope protein Nesprin-1. Nesprins, a family of proteins of the LINC complex that links the nuclear envelope and skeleton to the cytoskeleton, are believed to influence nuclear architecture and position as well as cytoskeletal stability and cell mobility. Nesprin-2 has been reported to bind MAPK in smooth muscle cells. The increased expression of Nesprin-1 in ES cells differentiated to primitive endoderm cells may therefore function to restrict MAPK nuclear entry. The limited expression of Nesprin-1 in undifferentiated ES cells would be permissive for MAPK nuclear entry and, as a consequence, enhance proliferation. We will test this hypothesis in the following three experimental aims: (1) Characterize Nesprin- 1/MAPK association biochemically; (2) Analyze Nesprin-1 and MAPK association in ES cell differentiation; and (3) Determine the impact of Nesprin-1 suppression on MAPK nuclear entry and signaling and cellular differentiation and proliferation. Control of cell growth and proliferation is an obligate step to attain and maintain the phenotype and function of differentiated cells in the developing embryo and in the adult organism, and requires regulation of MAPK cytoplasmic localization and nuclear entry. If the experiments suggest the hypothesis is valid, we may uncover a mechanism regulating MAPK localization and cell proliferation in differentiated cells. We speculate that regulation of nuclear entry of activated MAPK is prevalent in vivo, and this regulatory step is overcome in most cultured cells. PUBLIC HEALTH RELEVANCE: In this proposal we plan to investigate a possible mechanism for the restriction of nuclear entry of activated MAPK in differentiated ES cells. We suggest that this restriction is a prevalent, important regulatory step in vivo, which is required to maintain the differentiated phenotype. Moreover, it may be lost upon adaptation of cells in culture. Thus, this study may have the potential to uncover a regulatory mechanism for the Ras/MAPK signaling pathway that is highly critical in the regulation of cell growth, differentiation, and function.